Recombinant prothrombin analogues and uses thereof

ABSTRACT

During the process of coagulation, prothrombin is activated to α-thrombin by prothrombinase. Key residues in the structure of prothrombin allow for modulation of the activation of prothrombin. In certain embodiments, a recombinant prothrombin with at least one point mutation or deletion is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage Entry of PCT/US16/56049,filed on Oct. 7, 2016, which claims priority to and any benefit of U.S.Provisional Application No. 62/239,535, filed Oct. 9, 2015, the entirecontents of which are incorporated herein by reference in theirentirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named27433.04088-1_ST25.txt, which is 67 kb in size, was created on Jan. 10,2019 and electronically submitted via EFS-web, is incorporated byreference in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to the field of bloodcoagulation. More particularly, the present disclosure relates tomodified recombinant prothrombin analogues and uses thereof.

BACKGROUND

During the final stages of blood coagulation, prothrombin (Pro) isconverted to the serine protease thrombin, an enzyme which plays acentral role in clot formation. Activation of prothrombin to thrombinresults from the proteolytic cleavage by factor Xa and factor Va.

There are instances wherein it is advantageous to short-circuit thenormal clotting mechanisms, or to modify overactive clotting mechanismsin an individual. For example, thrombosis is the formation of a bloodclot inside a blood vessel, obstructing the flow of blood through thecirculatory system. When a clot is significantly large enough to reducethe blood flow to a tissue, hypoxia can occur and metabolic productssuch as lactic acid can accumulate. A larger thrombus can cause a muchgreater obstruction to blood flow and may result in anoxia and, incertain circumstances, tissue death. There are also a number of otherconditions that can arise according to the location of the thrombus andthe organs affected. Conditions that put an individual at risk forthrombosis include, but are not limited to, pulmonary embolism,thrombophlebitis, deep vein thrombosis, arterial occlusion fromthrombosis or embolism, arterial reocclusion during or after angioplastyor thrombolysis, restenosis following arterial injury or invasivecardiological procedures, postoperative venous thrombosis or embolism,acute or chronic atherosclerosis, stroke, myocardial infarction, cancerand metastasis, and neurodegenerative diseases. However, currentanti-thrombotic therapies suffer from a number of drawbacks. Thus, itwould be desirable to provide an anti-coagulant agent which inhibitscoagulation with greater specificity and without the side effects ofcurrent therapies.

SUMMARY

While embodiments encompassing the general inventive concepts may takediverse forms, various embodiments will be described herein, with theunderstanding that the present disclosure is to be considered merelyexemplary, and the general inventive concepts are not intended to belimited to the disclosed embodiments.

The embodiments relate to modulation of the blood coagulation processincluding administration of modified forms of prothrombin (Pro) andtheir use as anti-coagulants. Unlike native or wild type prothrombin,the resulting modified-prothrombin molecule is highly stable. Thisstability is achieved by amino acid modifications that disrupt necessarycleavage sites so that they are no longer recognized by the specificproteases that initiate clot formation.

In an exemplary embodiment, a novel recombinant prothrombin (rPro)polypeptide is provided which is characterized by having anti-coagulantactivity and having the amino acid sequence of Pro with an amino acidmodification at a residue between 473 and 487. In certain exemplaryembodiments, at least one amino acid residue selected from Ser⁴⁷⁸,Leu⁴⁸⁰, and Gln⁴⁸¹ is modified.

In an exemplary embodiment, a method of inhibiting coagulation in asubject who has or is at risk of having thrombosis is provided. Themethod comprises administering the polypeptide(s) of the generalinventive concepts to a subject who has or is at risk of having arterialthrombosis.

In an exemplary embodiment, an isolated polynucleotide is provided whichencodes a polypeptide characterized by having anti-coagulant activityand having the amino acid sequence of prothrombin with amino acidmodification at a residue from 473 to 487.

In an exemplary embodiment, a pharmaceutical composition comprising arecombinant prothrombin in combination with pharmaceutically acceptablecarriers, vehicles, and/or adjuvants is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the pathways for Pro activation andseveral exemplary recombinant polypeptides according to variousembodiments of the general inventive concepts. Pro is converted to IIathrough two fXa-catalyzed cleavages at Arg²⁷¹ and at Arg³²⁰ resulting inIIa formation. The red rectangle denotes the fVa-independent site forfXa on Pro while the yellow rectangle represents the fVa-dependent sitefor fXa studied herein. The light blue rectangle denotes the amino acidscomposing (pro)exosite I. All mutants used in the study are showntogether with their assigned name used throughout the this disclosure.

FIG. 2 is a graph of the clotting activity of all forms of Pro. Theaverage clotting time found in four different measurements inPro-deficient plasma is shown for all Pro/rPro molecules identified atthe bottom of the graph.

FIG. 3 is an analyses of Pro^(PLASMA) and rPro^(Δ473-487) moleculesactivation by membrane-bound fXa alone or prothrombinase. Panel A:rPro^(PLASMA) (1.4 μM) in the presence of PCPS vesicles, DAPA, andmembrane bound fXa alone (5 nM); panel B: rPro^(Δ473-487) (1.4 μM) inthe presence of PCPS vesicles, DAPA, and membrane bound fXa alone (5nM). Panel C: rPro^(PLASMA) (1.4 μM) in the presence of PCPS vesicles,DAPA, and prothrombinase (1 nM fXa and 20 nM fVa); panel D:rPro^(Δ473-487) (1.4 μM) in the presence of PCPS vesicles, DAPA, andprothrombinase (1 nM fXa and 20 nM fVa). Aliquots were withdrawn atvarious time intervals and treated as described. M represents the lanewith molecular weight markers (from top to bottom): 98000, 64000, 50000,and 36000, respectively. Lanes 1-19 show samples from the reactionmixture before (0 min) the addition of fXa and 20 s, 40 s, 60 s, 80 s,100 s, 120 s, 150 s, 180 s, 210 s, 240 s, 5 min, 6 min, 10 min, 20 min,30 min, and 60 min, 90 min, and 120 min respectively, after the additionof fXa. Following scanning densitometry as described in the Examplessection, the data representing Pro consumption as a function of time(sec) were plotted using non-linear regression analysis according to theequation representing a first-order exponential decay and the rates ofPro consumption using the apparent first-order rate constant, k (s⁻¹)obtained directly from the fitted data, were calculated as described andare reported in Table 1. Pro derived fragments are identified to theright of panels A, B, C, and D as follows: Pro, Pro (amino acid residues1-579); P1, prethrombin-1 (amino acid residues 156-579); F1⋅2-A,fragment 1⋅2-A chain (amino acid residues 1-320); F1⋅2, fragment 1⋅2(amino acid residues 1-271); P2, prethrombin-2 (amino acid residues272-579); B, B chain of IIa (amino acid residues 321-579); F1, fragment1 (amino acid residues 1-155).

FIG. 4 is an analyses of rPro molecules activation by membrane-bound fXaalone. Panel A: rPro^(WT) (1.4 μM) in the presence of PCPS vesicles,DAPA, and membrane bound fXa alone (5 nM); panel B: rPro^(ΔS5V) (1.4 μM)in the presence of PCPS vesicles, DAPA, and membrane-bound fXa alone (5nM). Aliquots were withdrawn at various time intervals and treated asdescribed. M represents the lane with molecular weight markers (from topto bottom): 98000, 64000, 50000, and 36000, respectively. Lanes 1-19show samples from the reaction mixture before (0 min) the addition offXa and 20 s, 40 s, 60 s, 80 s, 100 s, 120 s, 150 s, 180 s, 210 s, 240s, 5 min, 6 min, 10 min, 20 min, 30 min, and 60 min, 90 min, and 120 minrespectively, after the addition of fXa. Panel C: the two gels shown inFIGS. 4A and 4B together with similar gels obtained with all rProstudied were scanned and rPro consumption was recorded as described inthe Examples section. Following scanning densitometry, and normalizationto the initial Pro concentration, the data representing rPro consumptionas a function of time (sec) were plotted using non-linear regressionanalysis according to the equation representing a first-orderexponential decay using the software Prizm (GraphPad, San Diego,Calif.). Prothrombinase was assembled with rPro^(WT) (filled circles; R²0.98), rPro^(ΔC10) (filled squares; R² 0.98), rPro^(ΔN10) (filledtriangles; R² 0.99), rPro^(ΔS5V) (filled inverse triangles; R² 0.99),rPro^(S478A) (filled diamonds; R² 0.99), rPro^(L480A) (open squares; R²0.99), rPro^(SQ→AA) (open circles; R² 0.98), rPro^(SL→AA) (opentriangles; R² 0.99), and rPro^(SLQ→AAA) (open inverse triangles; R²0.99). The rates of rPro consumption shown in panel C, using theapparent first-order rate constant, k (s⁻¹) obtained directly from thefitted data, were calculated as reported ( ) and the data is shown inTable 1. Panel D: Schematic representation of fragments derivedfollowing Pro activation by membrane-bound fXa alone. The red arrowindicates impaired cleavage at Arg³²⁰ in rPr^(ΔC10) (filled squares),rPro^(ΔN10) (filled triangles), rPro^(ΔS5V) (filled inverse triangles)and rPro^(SLQ→AAA) (open inverse triangles) resulting in prethrombin-2generation. Pro derived fragments are identified to the right of eachpanel, according to the description provided in the legend of FIG. 3.

FIG. 5 is an SDS-PAGE analyses of rPro molecules activation byprothrombinase. Panel A: rPro^(WT) (1.4 μM) in the presence of PCPSvesicles, DAPA, and prothrombinase (1 nM fXa and 20 nM fVa); panel B:rPro^(S478A) same conditions as panel in A; panel C: rPro^(ΔS5V) sameconditions as in panel A; panel D: rPro^(SLQ→AAA) same conditions as inpanel A. Aliquots were withdrawn at various time intervals and treatedas described. Same time points as described in the legend to FIG. 4. Proderived fragments are identified to the right of each panel, accordingto the description provided in the legend of FIG. 3.

FIG. 6 is an analyses of the rates of activation of rPro byprothrombinase. Panel A: the gels shown in FIG. 4 together with similargels obtained with all rPro studied, were scanned and rPro consumptionwas recorded as described in the Examples section. Following scanningdensitometry, the numbers were normalized to the initial concentrationof rPro studied, and the data representing rPro consumption as afunction of time (sec) were plotted using non-linear regression analysisaccording to the equation representing a first-order exponential decayusing the software Prizm (GraphPad, San Diego, Calif.). rPro^(WT)(filled circles; R² 0.98), rPro^(ΔC10) (filled squares; R² 0.99),rPro^(ΔN10) (filled triangles; R² 0.94), rPro^(ΔS5V) (filled inversetriangles; R² 0.99), rPro^(S478A) (filled diamonds; R² 0.97),rPro^(L480A) (open squares; R² 0.98), rPro^(SQ→AA) (open circles; R²0.99), rPro^(SL→AA) (open triangles; R² 0.99), and rPro^(SLQ→AAA) (openinverse triangles; R² 0.99). The inset shows the progress of thereaction during the first 180 s. The rates of rPro consumption using theapparent first-order rate constant, k (s⁻¹) obtained directly from thefitted data, were calculated as reported and shown in Table 1. Panel B:Schematic representation of fragments derived following rPro activationby prothrombinase in the presence of an excess fVa with respect to fXa.The red arrow indicates impaired cleavage (at Arg³²⁰) in rPr^(ΔC10)(filled squares), rPro^(ΔN10) (filled triangles), rPro^(ΔS5V) (filledinverse triangles) and rPro^(SLQ→AAA) (open inverse triangles).

FIG. 7 is a determination of kinetic parameters of prothrombinasecatalyzing cleavage and activation of various Pro molecules. IIageneration experiments were conducted as described in the Examples byvarying the substrate concentration and using a chromogenic substrate.The solid lines represent the nonlinear regression fit of the data usingPrizm GraphPad software according to the Henri Michaelis-Menten equation(V_(o)=V_(max)·[Pro]/K_(m)+[Pro]) to yield the K_(m) and k_(cat)(k_(cat)=V_(max)/E_(TOT), where E_(TOT) is the concentration of fullyassembled prothrombinase, in this case is 10 pM, Table 2).Prothrombinase activity with various rPro molecules is shown as follows:rPro^(WT) filled circles; Pro^(PLASMA) filled squares; rPro^(S478A)filled diamonds; rPro^(L480A) open squares; rPro^(SL→AA) open triangles;rPro^(SQ→AA) open circles; rPro^(SLQ→AAA) open inverse triangles.Kinetic constants reported in the text and in Table 2 were extracteddirectly from fitted data showed herein.

FIG. 8 shows the activation of plasma-derived fV by rIIa. Plasma-derivedfV (500 nM) was incubated with rIIa (4 nM) as described in the Examples.At selected time intervals, aliquots of the mixtures were removed, mixedwith 2% SDS, heated for 5 min at 90° C., and analyzed on a 4-12%SDS-PAGE followed by staining with Coomassie Blue. Lane 1 in all panelsdepicts aliquots of the mixture withdrawn from the reaction before theaddition of the isolated rIIa. Lanes 2-8 represent aliquots of thereaction mixture withdrawn at 10 min, 20 min, 30 min, 45 min, 60 min,120 min, and 180 min. The positions of all fV fragments are indicated atthe right of panels D and H. Fragments (a) and (b) of fV are identifiedas previously shown.

FIG. 9 shows the activation of recombinant fVIII by rIIa. rfVIII (500nM) was incubated with rIIa (4 nM) as described below. At selected timeintervals, aliquots of the mixtures were removed, mixed with 2% SDS,heated for 5 min at 90° C., and analyzed on a 4-12% SDS-PAGE followed bystaining with Coomassie Blue. Lane 1 in all panels depicts aliquots ofthe mixture withdrawn from the reaction before the addition of theisolated rIIa. Lanes 2-8 represent aliquots of the reaction mixturewithdrawn at 10 min, 20 min, 30 min, 45 min, 60 min, 120 min, and 180min. The positions of all rfVIII fragments are indicated at the right ofpanels D and H. Fragments from rfVIII are identified as previouslyshown.

FIG. 10 shows the activation of protein C (PC) by rIIa. Plasma-derivedPC (80 nM) was incubated with rIIa (8 nM), thrombomodulin, and PCPS asdescribed below. Following a 3-hr incubation period each individualsolution was mixed with 2% SDS, and 2% β-mercaptoethanol, heated for 5min at 90° C., and analyzed on a 4-12% SDS-PAGE followed by stainingwith Coomassie Blue. Lane 1 PC alone no IIa; lane 2 PC alone, no IIaincubated with buffer for 3 hr; lane 3, PC and rIIa^(ΔS5V); lane 4, PCand plasma-derived IIa; lane 5, PC and rIIa^(WT); lane 6, PC andrIIa^(S478A); lane 7, PC and rIIa^(L480A); lane 8, PC and rIIa^(SL→AA);lane 9, PC and rIIa^(SQ→AA); and lane 10, PC and rIIa^(SLQ→AAA).Positions of PC and APC heavy and light chains fragments are indicatedat the right (a/b heavy chains, and c light chain). The two heavy chainsof PC in plasma (a and b) have been identified earlier, differ by oneglycosylation site, and have been extensively studied.

FIG. 11 is a representation of the location of exosites in thestructures of prothrombin and thrombin. Representation of the highresolution crystal structures of Pro^(WT) (A) and IIa (B). A)Space-filling representation of human Pro. Residues Ser⁴⁷⁸, Leu⁴⁸⁰, andGln⁴⁸¹ are colored green; ABE-I and ABE-II residues are yellow and blue,respectively; the amino acids composing the catalytic triad are notsolvent-accessible and thus not visible. Other catalytic domain residuesare in light gray while those in fragment-1 and fragment-2 are in darkgray. B) Space-filling representation of IIa. Residues are colored asfor Pro with the addition of catalytic triad residues His³⁶³, Asp⁴¹⁹,and Ser⁵²⁵ (red) which are partially solvent-accessible. In parenthesesare the corresponding numbers according to the chymothrypsin numberingof IIa. Distances between the active Ser⁵²⁵ side chain hydroxyl andseveral other amino acids of interest are: 17 Å to Gln⁴⁸¹ OE1/NE2; 15 Åto Glu⁵⁴⁹ OE1; 17 Å to Arg³⁸² NH₂; 18 Å to Lys³⁸⁵ NH₂. The polar atomsat the end of the side chains were used as a reference because thesewould presumably be involved in intermolecular interactions.

DETAILED DESCRIPTION

Modification of prothrombin amino acid residues from 473 to 487, and incertain instances at least one amino acid residue selected from Ser⁴⁷⁸,Leu⁴⁸⁰, and Gln⁴⁸¹, allows for modulation of activity of the resultingpolypeptide. While not wishing to be bound by theory, it is believedthat by modifying the structure of a prothrombin polypeptide in residuesfrom 473 to 487, the polypeptide may be recognized by prothrombinase butis not cleaved into an active form for clot formation. In other words,the modified prothrombin occupies, but is cleaved at a reduced rate by,prothrombinase, thereby slowing the production of thrombin. Thus, themore stable modified form can slow the normal clot-forming processes byslowing (or preventing) native prothrombin from being activated byprothrombinase.

Preferred polypeptides according to the general inventive concepts aretherapeutic compounds that demonstrate anti-coagulant activity and maybe used as antithrombotic agents. The term “therapeutic compound” asused herein means any compound which can be used in therapy, whichimplies the curing of a disease or malfunction of the body and whichcovers prophylactic treatment.

In an exemplary embodiment, a novel recombinant prothrombin (Pro)polypeptide is provided which is characterized by having anti-coagulantactivity and having the amino acid sequence of Pro with amino acidmodifications at residues between 473 and 487. In certain exemplaryembodiments, at least one amino acid residue selected from Ser⁴⁷⁸Leu⁴⁸⁰, and Gln⁴⁸¹ is modified. The recombinant prothrombin polypeptideof claim 1, wherein the modification occurs in at least one of aminoacid residues Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹. In certain exemplaryembodiments, the modification is a deletion of at least one of aminoacid residues Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹. In certain exemplaryembodiments, the modification is a modification of amino acid residueSer⁴⁷⁸. In certain exemplary embodiments, the modification is amodification of amino acid residue Leu⁴⁸⁰. In certain exemplaryembodiments, the modification is a modification of amino acid residueGln⁴⁸¹. In certain exemplary embodiments, the modification is amodification of amino acid residues Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹.

In an exemplary embodiment, a method of inhibiting coagulation in asubject who has or is at risk of having thrombosis is provided. Themethod comprises administering the polypeptide(s) of the generalinventive concepts to a subject who has or is at risk of having arterialthrombosis.

In an exemplary embodiment, an isolated polynucleotide is provided whichencodes a polypeptide characterized by having anti-coagulant activityand having the amino acid sequence of prothrombin with amino acidmodification at a residue from 473 to 487.

Unlike native or wild type prothrombin, the resultingmodified-prothrombin molecule is highly stable. This stability isachieved by amino acid modifications that disrupt necessary cleavagesites so that they are no longer recognized by the specific proteasesthat otherwise initiate clot formation.

In an exemplary embodiment, a method of inhibiting coagulation in asubject who has, or is at risk of having, thrombosis is provided. Themethod comprises administering the polypeptide(s) of the generalinventive concepts to a subject who has, or is at risk of having,thrombosis.

In an exemplary embodiment, an isolated polynucleotide is provided whichencodes a polypeptide characterized by having anti-coagulant activityand having the amino acid sequence of prothrombin with amino acidmodification at a residue from 473 to 487.

In an exemplary embodiment, a pharmaceutical composition comprising arecombinant prothrombin in combination with pharmaceutically acceptablecarriers, vehicles, and/or adjuvants is provided.

The general inventive concepts also provide a method for amelioratingthrombosis in a subject having, or at risk of having, thrombosiscomprising administering a therapeutically effective amount of thepolypeptide(s) described herein. As used herein, the term “ameliorate”denotes a lessening of the detrimental effects of thrombosis in thesubject receiving therapy. The term “therapeutically effective” meansthat the amount of polypeptide used is of sufficient quantity toameliorate or modulate the effects of the thrombosis.

The term “anti-coagulant activity” as used herein, when referring to theinventive methods and compositions described herein, refers to reducedcoagulant or procoagulant activity relative to a wild-type or nativepolypeptide. In certain exemplary embodiments, the inventivepolypeptides demonstrate anti-coagulant activity that is reduced by atleast 10% relative to the native polypeptide (i.e., the activity isreduced by 10% to 100%). In certain exemplary embodiments, the inventivepolypeptides demonstrate anti-coagulant activity that is reduced by atleast 20% relative to the native polypeptide. In certain exemplaryembodiments, the inventive polypeptides demonstrate anti-coagulantactivity that is reduced by at least 40% relative to the nativepolypeptide. In certain exemplary embodiments, the inventivepolypeptides demonstrate anti-coagulant activity that is reduced by atleast 60% relative to the native polypeptide. In certain exemplaryembodiments, the inventive polypeptides demonstrate anti-coagulantactivity that is reduced by at least 80% relative to the nativepolypeptide. In certain exemplary embodiments, the inventivepolypeptides demonstrate anti-coagulant activity that is reduced by atleast 90% relative to the native polypeptide. In certain exemplaryembodiments, the inventive polypeptides demonstrate anti-coagulantactivity that is reduced by at least 95% relative to the nativepolypeptide. Anti-coagulant activity may be determined by methods suchas those described in the Examples section, including but not limited toin vitro determination.

The terms “susceptible” and “at risk” as used herein, unless otherwisespecified, mean having resistance to a certain condition or disease thatis reduced relative to the population as a whole, including beinggenetically predisposed, having a family history of, and/or havingsymptoms of the condition or disease.

The terms “modulating” or “modulation” or “modulate” as used herein,unless otherwise specified, refer to the targeted movement of a selectedcharacteristic.

The term “modification” as used herein, when referring to an amino acidsequence, unless otherwise specified, refers to a substitution of oneamino acid for another, including deletion of the original amino acid.

The polypeptides of the general inventive concepts are a derivative of anaturally occurring physiologic human protein. Due to their specificityof action and dosage requirements, the polypeptides of the generalinventive concepts may prove superior to other available antithromboticagents in terms of at least one of bleeding tendency, toxicity,antigenicity, clearance rate, general side effects, and allergicreactions. Therefore, administration of the polypeptides of the generalinventive concepts, alone or in combination with other thrombolytic orfibrinolytic agents, may prove useful in various clinical situations.

Minor modifications of the primary amino acid sequence of thepolypeptides of the general inventive concepts may result inpolypeptides which have substantially equivalent activity as compared tothe specific polypeptides described herein. Such modifications may bedeliberate, as by site-directed mutagenesis, or may be spontaneous. Allof the polypeptides produced by these modifications are included hereinas long as the biological activity of the inventive polypeptide stillexists. For example, a modified polypeptide must still contain thecleavage sites which cannot be recognized by the specific protease.Further, deletion of one or more amino acids can also result in amodification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would also have utility.For example, it may be possible to remove amino or carboxy terminalamino acids which may not be required for biological activity of theparticular polypeptide.

In addition to the discrete proteolytic sites described herein, theexemplary embodiments embrace conservative variations in the remainingamino acid sequence of the polypeptide of this disclosure. The term“conservative variation” as used herein denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term does not refer to thespecific cleavage sites indicated herein.

The exemplary embodiments also relate to polynucleotides which encodethe polypeptides of the general inventive concepts. As used herein,“polynucleotide” refers to a polymer of deoxyribonucleotides orribonucleotides, in the form of a separate fragment or as a component ofa larger construct. DNA encoding a peptide of the general inventiveconcepts can be assembled from cDNA fragments or from oligonucleotideswhich provide a synthetic gene which is capable of being expressed in arecombinant transcriptional unit. Polynucleotide sequences of thegeneral inventive concepts include DNA, RNA, and cDNA sequences. Apolynucleotide sequence can be deduced from the genetic code, however,the degeneracy of the code must be taken into account. Polynucleotidesof the general inventive concepts include sequences which are degenerateas a result of the genetic code.

In certain embodiments, the relevant polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus, or other vehicle known inthe art that has been manipulated by insertion or incorporation of thegenetic sequences. Such expression vectors contain a promoter sequencewhich facilitates the efficient transcription of the inserted geneticsequence of the host. The expression vector typically contains an originof replication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells.

The exemplary embodiments also provide a method for producing apolypeptide which acts as a prothrombin molecule which is activated at areduced rate, including polypeptides which cannot be activated. Themethod includes the steps of introducing into a host cell an expressionvector which contains a nucleotide sequence which encodes a polypeptidewhich acts as an inactive prothrombin molecule and is incapable ofactivation; culturing the host cell in an appropriate medium; andisolating the polypeptide product encoded by the expression vector.

Based on the foregoing, because some of the method embodiments of thepresent disclosure are directed to specific subsets or subclasses ofidentified individuals (that is, the subset or subclass of individuals“in need” of assistance in addressing one or more specific diseases orspecific conditions noted herein), not all individuals will fall withinthe subset or subclass of individuals as described herein for certaindiseases or conditions.

In the presence of a procoagulant membrane surface and divalent metalions, factor Va (fVa) binds factor Xa (fXa) to form prothrombinase.Prothrombinase is the two subunit enzymatic complex where thenon-enzymatic regulatory subunit (fVa) controls the rate and directscleavage of Pro by the catalytic subunit (fXa) at two spatially distinctsites resulting in timely α-thrombin (IIa) formation at the place ofvascular injury. Cleavage at Arg²⁷¹ and Arg³²⁰ of Pro is required toform the active serine protease IIa. The essential IIa molecule bearsstrong homology with other serine protease enzymes, such as activatedprotein C (APC), chymotrypsin, and fXa. Several different numberings ofIIa residues appear in the literature based on either the chymotrypsinnumbering, or IIa numbering, or the entire Pro sequence. The latternomenclature is used herein with the appropriate chymotrypsin numberingin parenthesis when required for comparison with the existingliterature.

Historically, it has been shown that in the absence of fVa, initialcleavage at Arg²⁷¹ of Pro results in the generation of the inactiveintermediate prethrombin-2 and fragment 1⋅2. Further cleavage ofprethrombin-2 at Arg³²⁰ generates IIa (prethrombin-2 pathway) (FIG. 1).Concurrent with the appearance of excess fVa during clotting, the orderof cleavages is reversed, and initial cleavage at Arg³²⁰ generates atransient enzymatically active intermediate, meizothrombin, that hasmuch higher catalytic efficiency than IIa towards chromogenic substratesusually employed to assess IIa activity. Meizothrombin is cleaved atArg²⁷¹ resulting in the generation of IIa and fragment 1⋅2(meizothrombin pathway). While efficient cleavage at each site requiresthe presence of phospholipids, initial cleavage at Arg³²⁰ is entirelyfVa-dependent.

In the absence of fVa, Pro is activated at a slow non-physiological rateby membrane-bound fXa alone. Interactions between fXa and Pro are knownto exist in the presence and absence of fVa, however, the enhancedactivity of fXa within prothrombinase is controlled solely by thenon-enzymatic cofactor. Consequently, the innate process of coagulationrests on specific molecular interactions involved in the fVa-dependentactivation of Pro by prothrombinase. In relation to fXa alone, therelative rate of IIa formation by prothrombinase is increased by300,000-fold because of the increase in the rates of both Pro cleavages.This increase is mainly associated with a large (3,000-fold) increase inthe k_(cat) of fXa within prothrombinase with a modest 100-fold decreasein the K_(m) of the enzyme. This substantial increase in enzymaticactivity resulting in rapid IIa generation is credited through preciseand unique interactions of the cofactor with specific amino acidsaffiliated with both membrane-bound fXa and membrane-bound Pro asrecently demonstrated. Accordingly, the introduction of thenon-enzymatic cofactor into prothrombinase equips the organism'scoagulation artillery necessary for the explosive arrest of vasculaturebleeding.

Factor V (fV) is a large quiescent multi-domain (A1-A2-B-A3-C1-C2)protein that circulates in blood at a concentration of 20 nM. Threesequential cleavages of fV at Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵ by IIa and/orfXa release the B domain and promote formation of the active cofactor,fVa. Pro circulates abundantly in blood at a concentration of 1.4 μM asthe zymogen form of the serine protease IIa. Mature Pro protein iscomposed of a region containing several post-translationally modifiedγ-carboxyglutamic acid residues (described as the Gla domain, residues1-46), followed by two kringle domains (residues 65-143 and 170-248,respectively) and a serine protease domain (residues 272-579, FIG. 1).Pro contains three linkers: linker 1 (residues 47-64) connects the Gladomain to kringle-1, linker 2 (residues 144-169) connects the twokringles, and linker 3 (residues 249-284) connects kringle-2 to theA-chain portion of IIa.

The necessary fVa-dependent activation of Pro by prothrombinase is awidely studied mechanism of coagulation but still poorly understood.Numerous fVa binding sites are acknowledged to exist on Pro. Earlierinvestigations have showed the existence of binding sites on Pro for fVain each of the kringle domains, and within the Gla domain. Furthermore,significant protein-protein interactions between the acidicCOOH-terminal region of fVa and a region rich in basic amino acids ofPro have been inferred and characterized indirectly by employingmolecular techniques involving specific hirudin-like ligands and theanion binding (pro)exosite I (ABE I) of Pro derivatives, as well asdirectly using a specific acidic peptide derived from the COOH-terminalregion of the fVa heavy chain and recombinant fVa molecules.Site-directed mutagenesis of these basic residues generated arecombinant prothrombin molecule impaired in its ability to fullyinteract with fVa during complex formation. While a crystal structureand a model of fVa have been available for some time now, the crucialinteraction of the acidic hirudin-like COOH-terminal portion of theheavy chain of the cofactor with Pro required for efficient IIaformation was initially ignored because it was missing from the crystalstructure of the cofactor. This interaction was further discountedwithout providing any solid evidence in spite of initial findings byGuinto and Esmon and more recent original findings from our laboratory.A very recent model of prothrombinase, using as a template the crystalstructure of prothrombinase from the snake venom of Pseudonaja textilis,verified and established the critical role of the acidic COOH-terminalregion of fVa heavy chain for timely Pro cleavage and activation at twospatially distinct sites by prothrombinase.

Additional studies with several recombinant prethrombin-1 molecules,where seven critical basic amino acids within (pro)exosite 1 werechanged to glutamic acid confirmed the interaction of (pro)exosite Iwith fVa acidic regions. Notably, the data revealed that while mutatedprethrombin-1 is a poor substrate for prothrombinase, the same moleculewas activated by membrane-bound fXa alone with similar rates as wildtype prethrombin-1. Supplementary to these studies, Yegneswaran et al.,utilizing synthetic peptides derived from a highly conserved region ofPro, postulated the existence of a fVa-dependent binding exosite for fXawithin the sequence 473-487 (chymotrypsin numbering 149D-163) of Prothat is in close spatial arrangement to the (pro)exosite I. Although theauthors suggested an important scaffold for a fVa-dependent bindingexosite for fXa on Pro, their peptide studies discounted the importanceof a minimal but significant stretch of amino acids within the regioncomposed of amino acids 478-482 (Ser-Val-Leu-Gln-Val, chymotrypsinnumbering 153-157) of Pro. The same authors have also identified afVa-independent site for fXa on prothrombin (amino acids 557-571).

The study described below was initiated in order to identify andinvestigate the identity and role of the minimum important amino acidswithin the stretch 478-482 of Pro that is conserved in a wide-range ofmammalian species and regulates peptide bond specificity and Proactivation by prothrombinase in a fVa-dependent manner. Our unexpectedand original findings provide evidence that specific amino acids withinPro have a dual role in providing for both, a fVa-dependent exosite forfXa for efficient Pro activation required for timely cleavage at Arg³²⁰,while also serving as the newly defined important exosite for proper andefficient tethering of IIa's physiological substrates, which in turn isrequired for optimum physiological IIa activity.

EXAMPLES

The following examples illustrate specific embodiments and/or featuresof the general inventive concepts. The examples are given solely for thepurpose of illustration and are not to be construed as limitations ofthe present disclosure, as many variations thereof are possible withoutdeparting from the spirit and scope of the disclosure.

Materials.

Phenylmethylsulfonylfluoride (PMSF), O-phenylenediamine (OPD)dihydrochloride, N-[2-Hydroxyethyl]piperazine-N′-2-ethanesufonic acid(Hepes), Trizma (Tris base), and Coomasie Blue R-250 were purchased fromSigma (St. Louis, Mo.). fV-deficient plasma was purchased from ResearchProtein Inc. (Essex Junction, Vt.). Secondary anti-mouse, anti-sheep,and anti-equine IgG coupled to peroxidase were from SouthernBiotechnology Associates Inc. (Birmingham, Ala.). L-α-phosphatidylserine(PS) and L-α-phosphatidylcholine (PC) were from Avanti Polar Lipids(Alabaster, Ala.). Chemiluminescent reagent ECL Plus, Heparin-Sepharose,MonoQ 5/50 columns were from GE Healthcare Life Sciences (Pittsburgh,Pa.). Normal reference plasma and chromogenic substrate Spectrozyme-THwere from American Diagnostica Inc. (Greenwich, Conn.). S-2238 was fromAnaSpec (Fremont, Calif.) and recombiPlasTin used in the clotting assayswas purchased from Instrumentation Laboratory Co (Lexington, Mass.). Thereversible fluorescent IIa inhibitordansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), humanplasma-derived PC human-plasma-derived IIa, human plasma-derived Pro,and Pro-deficient plasma were purchased from Haematologic TechnologiesInc. (Essex Junction, Vt.). The purified human plasma-derived protein C(PC) preparation used contained both heavy chains isoforms that areactivated to activated protein C (APC) with similar rates as describedearlier. Human fXa was purchased from Enzyme Research Laboratories(South Bend, Ind.). The plasmid pZEM229R-lite encoding human recombinantprothrombin (rPro) was a generous gift from Dr. Kathleen Berkner(Cleveland Clinic Foundation, Cleveland, Ohio). QuikChange® II XL SiteDirected Mutagenesis Kit was obtained from Agilent Technologies Genomics(Santa Clara, Calif.). All molecular biology and tissue culturereagents, specific primers, and medium were obtained from Gibco,Invitrogen Corp. (Grand Island, N.Y.) or as indicated. Monoclonalantibodies to fV (αHFV_(HC)17 and αHFV_(LC)9), monoclonal antibody αHFV1coupled to Sepharose used to purify plasma and recombinant fV molecules,and a polyclonal antibody to Pro used for immunoblotting experimentsduring rPro production were provided by Dr. Kenneth G. Mann (Departmentof Biochemistry, University of Vermont, Burlington, Vt.). Plasma factorV (fV^(PLASMA)) and plasma fVa (fVa^(PLASMA)) were purified aspreviously described.

Construction of rPro Molecules.

To investigate the importance of amino acid region 473-487 of the serineprotease domain of Pro, we first constructed a recombinant mutant Promolecule with this region deleted (rPro^(Δ473-487)) using Stratagene'sQuikChange® site-directed mutagenesis kit and the pZEM229R-lite plasmid.rPro^(Δ473-487) was constructed using the mutagenic primers 5′-GAG ACGTGG ACA GCC AAC GTT GTG GAG CGG CCG GTC TGC AAG-3′ (sense) and 5′-CTTGCA GAC CGG CCG CTC CAC AAC GTT GGC TGT CCA CGT CTC-3′ (antisense)(corresponding to the ⁴⁷³GKGQPSVLQVVNLPI⁴⁸⁷ deletion). The mutation wasconfirmed by DNA sequencing (DNA Analysis Facility, Department ofMolecular Cardiology at The Learner Research Institute, ClevelandClinic, Cleveland Ohio).

To further investigate the minimum sequence of amino acids required forthe fVa-dependent fXa binding on Pro within the region 473-487 of theserine protease domain, several rPro molecules with the mutationsdenoted as rPro^(ΔN10), rPro^(ΔC10), rPro^(ΔS5V), rPro^(S478A),rPro^(L480A), rPro^(SL→AA), rPro^(SQ→AA) and rPro^(SLQ→AAA) (FIG. 1)were constructed using Stratagene's QuikChange® Site-DirectedMutagenesis Kit and the pZEM229R-lite plasmid. First, overlappingdeletions in the region 473-487 were constructed using the mutagenicprimers for rPro^(ΔN10) 5′-G ACG TGG ACA GCC AAC GTT GTG AAC CTG CCC ATTGTG GAG-3′ (sense) and 5′-CTC CAC AAT GGG CAG GTT CAC AAC GTT GGC TGTCCA CGT C-3′ (antisense) (corresponding to the ⁴⁷³GKGQPSVLQV⁴⁸²deletion), while mutagenic primers used for rPro^(ΔC10) were 5′-GTT GGTAAG GGG CAG CCC GTG GAG CGG CCG GTC TGC-3′ (sense) and 5′-GCA GAC CGGCCG CTC CAC GGG CTG CCC CTT ACC AAC-3′ (antisense) (corresponding to the⁴⁷⁸SVLQVVNLPI⁴⁸⁷ deletion). Similarly, the middle deletion of theoverlapping mutations rPro^(ΔS5V) was constructed using the mutagenicprimers 5′-GTT GGT AAG GGG CAG CCC GTG AAC CTG CCC ATT GTG-3′ (sense)and 5′-CAC AAT GGG CAG GTT CAC GGG CTG CCC CTT ACC AAC-3′ (antisense)(corresponding to the ⁴⁷⁸SVLQV⁴⁸² middle deletion). Next, within thesequence 478-482, several point mutations were made based on amino acidsolvent exposure and homology within other proteins. The first rPropoint alanine mutation rPro^(S478A) was constructed using the mutagenicprimers 5′-GGT AAG GGG CAG CCC GCA GTC CTG CAG GTG-3′ (sense) and 5′-CACCTG CAG GAC TGC GGG CTG CCC CTT ACC-3′ (antisense) (corresponding to theSer⁴⁷⁸→Ala mutation). The next single point mutation rPro^(L480A) wasconstructed using the mutagenic primers 5′-GGG CAG CCC AGT GTC GCG CAGGTG GTG AAC CTG CCC-3′ (sense) and 5′-GGG CAG GTT CAC CAC CTG CGC GACACT GGG CTG CCC-3′ (antisense) (corresponding to the L⁴⁸⁰→A mutation).In addition to single point mutations, we constructed two double alaninemutations, rPro^(SL→AA) and rPro^(SQ→AA), within the stretch 478-482 ofPro. For rPro^(SL→AA) we used the mutagenic primers 5′-GGT AAG GGG CAGCCC GCA GTC GCG CAG GTG GTG AAC CTG-3′ (sense) and 5′-CAG GTT CAC CACCTG CGC CAC TGC GGG CTG CCC CTT ACC-3′ (antisense) (corresponding to theSer⁴⁷⁸/Leu⁴⁸⁰→AA mutation). Also, for rPro^(SQ→AA), we used themutagenic primers 5′-GGT AAG GGG CAG CCC GCA GTC CTG GCG GTG GTG AACCTG-3′ (sense) and 5′-CAG GTT CAC CAC CGC CAG GAC TGC GGG CTG CCC CTTACC-3′ (antisense) (corresponding to the Ser⁴⁷⁸/Gln⁴⁸¹→AA mutation).Lastly, we constructed a Pro molecule with a triple mutation,rPro^(SLQ→AAA) with the mutagenic primers 5′-GGT AAG GGG CAG CCC GCA GTCGCG GCG GTG GTG AAC CTG-3′ (sense) and 5′-CAG GTT CAC CAC CGC CGC GACTGC GGG CTG CCC CTT ACC-3′ (antisense) (corresponding to theSer⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹→AAA mutation) and a rPro molecule withSer⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹ deleted (rPro^(ΔSLQ)) using the mutagenic primers:5′-GGT AAG GGG CAG CCC GTC GTG GTG AAC CTG CCC-3′ (sense) and 5′-GGG CAGGTT CAC CAC GAC GGG CTG CCC CTT ACC-3′ (antisense). All deletions andpoint mutations were confirmed by DNA sequencing (DNA Analysis Facility,Department of Molecular Cardiology at The Learner Research Institute,Cleveland Clinic, Cleveland Ohio).

Expression of Wild-Type and Mutant rPro Molecules in Mammalian Cells.

rPro expression in baby hamster kidney (BHK-21) cells has beenpreviously described in detail. Briefly, BHK-21 cells were maintained inDulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovineserum (10%) and a streptomycin/penicillin (1%) mixture. Isolatedplasmids (4-6 μg) for wild-type and mutant rPro molecules weretransfected into the BHK-21 cells using a lipid based transfectionreagent, lipofectamine (Invitrogen Corp), according to themanufacturer's instructions. Following an incubation period of 48 h,DMEM was supplemented with fetal bovine serum (10%),streptomycin/penicillin (1%) mixture, and methotrexate (1 μM) and addedto the cells. After three weeks of treatment with the selection medium,colonies were isolated, grown, and screened for levels of Pro expressionby western blot analysis and compared to plasma-derived Pro as astandard (1 μg/ml). Identification of the highest secreting rPro clonewas further used in large-scale protein expression with serum-freeOpti-MEM supplemented with ZnCl₂ (50 μM), vitamin K₁ (10 μg/ml), andpenicillin/streptomycin/Fungizone (1% v/v) mixture, and the medium wascollected every 2 days for 2-3 weeks. Following collections, the mediawas stored at −80° C. until the desired amount (usually 4 L) wasobtained and used for purification.

Purification of rPro Molecules.

Purification of rPro was performed through a well-established protocolpreviously described in detail. Briefly, collected media was thawed,filtered (0.45 μm), and loaded on a tandem column setup of amberliteXAD₂ and Q-Sepharose. Following the complete addition of medium to thetwo columns, the Q-Sepharose column was separated and washed with TBS(0.02 M Tris, 150 mM NaCl, pH 7.4). The bound material containing rProon the Q-Sepharose was eluted with 0.02 M Tris, 0.5 M NaCl, and pH 7.4.The material was treated with barium citrate, and the isolated pelletwas dissolved in a minimum volume of EDTA (0.5 M, pH 7.4). The dissolvedpellet was dialyzed twice in fresh TBS (2×4 L) and filtered (0.45 m)prior to being loaded onto an General Electric (GE) Fast PerformanceLiquid Chromatography (FPLC) instrument, equipped with a strong anionicexchanger MonoQ 5/50 column. The column was equilibrated in TBS, and astep-wise gradient of calcium (0-50 mM) in TBS was used to isolate fullygamma-carboxylated rPro. Tubes containing the rPro molecules wereconcentrated using an appropriate Millipore Centricon (Billerica,Mass.), and aliquots were frozen at −80° C. to avoid repeatedfreeze-thaw cycles. Following purification and before any experiment,all rPro molecules were characterized as extensively described below.

The level of γ-carboxylation of all rPro molecules was determinedfollowing alkaline hydrolysis coupled to amino acid analysis performedat the Texas A&M University protein chemistry facility as described. Allpurified molecules were found to be properly carboxylated (Table 1). Toverify that rPro^(WT) and rPro^(Δ473-487) are processed at theappropriate cleavage sites when incubated with prothrombinase or fXaalone and produce the expected fragments, the recombinant proteins wereincubated with PCPS vesicles and fXa in the presence and absence of fVa.Following gel electrophoresis, fragments were transferred to PVDFmembrane and identified following NH₂-terminal sequencing. All fragmentsderiving from the recombinant prothrombin molecules have the expectedNH₂-terminal sequence following cleavage by either prothrombinase ormembrane-bound fXa alone (not shown).

The fact that the rPro^(Δ473-487) molecule contains a fifteen amino aciddeletion was verified by cDNA sequencing. However, in view of thesurprising and unexpected data presented herein, it was important toconfirm the existence of the deletion in the purified recombinantprotein. This was accomplished by Mass Spectrometry. Briefly, followingactivation of rPro and Pro^(PLASMA) by prothrombinase, aliquots wereanalyzed in triplicated under reducing conditions on a 12% SDS-PAGE.Following staining/destaining, the B-chain of IIa was excised from thegel, and the proteins were reduced and alkylated with iodoacetamide.Digestion (in gel) was accomplished with Porcine Typsin. Analysis of theresulting peptides was performed with an alpha cyanohydroxycinnamic acid(matrix) Kratos Axima CFR MALDI-TOF mass spectrometer (reflector mode;25000 accelerating voltage) in the Protein Chemistry Laboratory at TexasA & M University under the direction of Dr. Larry Dangott. The dataverified the existence of the deletion in rPro^(Δ473-487) Similarexperimental work demonstrate that all rPro molecules described hereinare fully carboxylated, can be appropriately processed by prothrombinaseand fNa alone, and do indeed contain the expected deletion/mutation s.

Gel Electrophoresis and Western Blotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed according to the method of Laemmli, using 9.5% gels followingreduction with 2% β-mercaptoethanol. Screening for high levels of rProsecreting clones was performed by western blotting using polyvinylidenediflouride (PVDF) according to Towbin et al. Successfully transferredproteins were visualized by chemiluminescence using ECL Plus reagentsfollowing incubation with a polyclonal antibody specific toprethrombin-1.

Studies of the Pathway for Pro Activation by Gel Electrophoresis.

The investigation of the activation rates of plasma-derived and of allrPro molecules cleavage and activation by fXa alone or prothrombinasewas performed according to a protocol described by our laboratory inmany instances using plasma-derived Pro or rPro. The calculation of therates of all Pro molecules consumption by fXa alone or by prothrombinasewere performed as previously described with the software Prizm.

Kinetic Titrations of Prothrombinase.

To investigate the kinetic constants (K_(m) and k_(cat)) ofprothrombinase, assays with a set amount of plasma-derived fVa and fXa(as described in the legend to the figures) were executed as describedby our laboratory in many instances. Each experiment used to reportfinal numbers was run at least in duplicate, and the goodness of fit(R²) for every model tested is provided in the results section. Theinitial rate of IIa generation was analyzed with the software Prizm(GraphPad), and all final numbers reported are derived directly from thegraphs. The change in transition-state stabilization free energy, whichmeasures the effect of the mutations in rPro was calculated for thedouble and triple mutants as extensively detailed in the literature andrepetitively reported by our laboratory.

Recombinant Thrombin Activity.

rPro molecules were converted to rIIa by 1 nM prothrombinase. Thechromogenic substrate S-2238 was used to assess rIIa activity byemploying serial dilutions in Tris-NaCl buffer in the presence of 0.1%PEG 8000. The final concentrations of S-2238 used in the reactions were0.94 μM, 1.87 μM, 3.75M, 7.50 μM, 15 μM, and 60 μM. The reaction wasstarted by the addition of 4 nM rIIa. The data was obtained at 1 minusing a SpectraMax M2 Platereader (Molecular Devices). The opticaldensity was automatically adjusted for a 1 cm pathlength, and theV_(max) was calculated from the optical density using the establishedextinction coefficient of S-2238 at room temperature following plottingof the data to the Michaelis-Menten equation using the software Prizm.

Activation of fV and fVIII by rIIa.

rPro molecules were converted to rIIa by 1 nM prothrombinase. Theresulting wild-type and mutant IIa were assessed in their ability tocleave and activate the cofactors over time by SDS-PAGE. Reactionmixtures containing either 500 nM plasma derived human fV or recombinanthuman fVIII were diluted in Tris-NaCl buffer in the presence of Ca²⁺.The final concentration of rIIa in the mixtures was 4 nM.

Activation of protein C by plasma-derived IIa or rIIa. rPro moleculesdescribed herein were converted to IIa by 1 nM prothrombinase. Theresulting IIa molecules were assessed for their ability to cleave andactivate PC in the presence of thrombomodulin and PCPS vesiclesaccording to a procedure previously described in Tris-buffered salinewith Ca²⁺. PC activation was verified by SDS-PAGE under reducingconditions. The final concentration of IIa in all mixtures was 8 nM.Gels were stained with Coomassie Brilliant Blue.

Pro Clotting Assay.

To assess the function of all Pro molecules in whole plasma, a clottingassay using Pro-deficient plasma was employed. The clotting assay wasperformed as previously described, and the time needed for formation ofa fibrin clot was monitored at 37° C. using a Diagnostica Stago STart® 4Hemostasis Analyzer as previously described.

Structural Analysis.

To evaluate the structural features of the Ser⁴⁷⁸, Leu⁴⁸⁰ and Gln⁴⁸¹residues, crystal structures of Pro and IIa were superimposed andcompared. The three human Pro crystal structures that have been reportedshow similar conformations for the residues of interest and neighboringregions; the highest resolution of these structures was chosen fordetailed analysis. From the many human IIa crystal structures that areavailable, several representative examples in different bound stateswere compared and found to have similar conformations for the regioncontaining the residues of interest. A high resolution structure ofunbound IIa was chosen as the representative structure for detailedanalysis. The program COOT was used to inspect structural features anddetermine distances. AREAIMOL was used to calculate thesolvent-accessible surface areas for specific residues, and molecularfigures were prepared with the PyMOL Molecular Graphics System, Version1.5.0.4 (Schrödinger, LLC).

rPro Expression.

To evaluate the minimal amino acid sequence necessary for thefVa-dependent Pro activation by prothrombinase within the recentlyidentified 473-487 critical amino acid stretch, we stably transfectedBHK-21 cell lines according to a previously defined protocol. rPro^(WT)and mutant rPro molecules as follows: rPro^(Δ473487) (missing residues⁴⁷³GKGQPSVLQVVNLPI⁴⁸⁷), rPro^(ΔN10) (missing amino acid residues⁴⁷³GKGQPSVLQV⁴⁸²), and rPro^(ΔC10) (missing amino acid residues⁴⁷⁸SVLQVVNLPI⁴⁸⁷) (FIG. 1). These three mutant molecules have five aminoacids in common (⁴⁷⁸SVLQV⁴⁸²). We have thus proceeded to construct,stably express, and purify to homogeneity rPro^(ΔS5V) (a rPro moleculemissing amino acids ⁴⁷⁸SVLQV⁴⁸²) (FIG. 1). Preliminary experiments withrPro^(ΔS5V) demonstrated that the mutant molecule had no clottingactivity as rPro^(Δ473487), rPro^(ΔN10) and rPro^(ΔC10). We nextproceeded to make single, double and triple alanine substitutions withinthis significant region. The following recombinant mutant molecules weremade: rPro^(S478A) (Ser⁴⁷⁸→Ala), rPro^(L480A) (Leu⁴⁸⁰→Ala), rPro^(SL→AA)(Ser⁴⁷⁸/Leu⁴⁸⁰→Ala), rPro^(SQ→AA) (Ser⁴⁷⁸/Gln⁴⁸¹→Ala) and rPro^(SLQ→AAA)(Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹→Ala). It is important to note that rIIa^(S478A)was previously tested and found to behave as rIIa^(WT). We have thusused this mutation as an internal control for all double and triplealanine substitutions of rPro. Finally, and to verify the resultsobtained with rPro^(SLQ→AAA) we have also made a rPro molecule withthese three amino acids deleted (rPro^(ΔSLQ)). In all experimentsresults with the mutant molecules were compared with results obtainedwith rPro^(WT) or Pro^(PLASMA).

Pro Times.

The ability of all rPro molecules to be activated under physiologicalconditions and promote fibrin clot formation was first assessed usingPro times (PTs) (FIG. 2).

The physical properties, clotting times, and rate of cleavage of variousrPro molecules in the presence of a fixed concentration ofmembrane-bound fXa or in the presence of prothrombinase are shown inTable 1.

TABLE 1 Rate of Pro Rate of Pro molecules molecules cleavage cleavage bymembrane- by prothrombinase^(c) mol Gla/mol Clotting time^(a) bound fXaalone^(c) (nM consumed · s⁻¹ · Mutant protein (s) (nM consumed · s⁻¹ ·fXa⁻¹) fXa⁻¹) Pro^(PLASMA)   10 ± 1 12.7 ± 0.12 0.13 ± 0.03 (0.94)^(d)17.0 ± 1.9 (0.99)^(d) rPro^(WT)   9.1 ± 0.9 12.2 ± 0.16 0.1 ± 0.012(0.99) 22.8 ± 3.8 (0.98) rPro^(Δ473-487)   9.5 ± 0.1 >120^(b) 0.53 ±0.013 (0.99) 1.0 ± 0.26 (0.92) rPro^(ΔN10) 12.0 ± 1 >120^(b) 0.7 ± 0.03(0.98) 1.5 ± 0.34 (0.94) rPro^(ΔC10) 12.5 ± 1 >120^(b) 0.35 ± 0.04(0.99) 1.3 ± 0.09 (0.99) rPro^(ΔS5V)   11 ± 1 >120^(b) 0.8 ± 0.03 (0.99)1.8 ± 0.12 (0.99) rPro^(S478A) 10.8 ± 1 11.75 ± 0.32  0.25 ± 0.015(0.99) 24.9 ± 4.6 (0.97) rPro^(L480A) 10.1 ± 1 29.8 ± 0.41 0.6 ± 0.02(0.99) 28.8 ± 2.8 (0.98) rPro^(SL→AA)   9.5 ± 0.9 28.7 ± 0.35 0.63 ±0.03 (0.99) 18.2 ± 3.7 (0.99) rPro^(SQ→AA)   10 ± 1 23.2 ± 0.3  0.23 ±0.025 (0.98) 35.9 ± 3.8 (0.99) rPro^(SLQ→AAA) 10.6 ± 1 116.2 ± 0.65  0.6± 0.04 (0.99) 1.23 ± 0.12 (0.98) rPro^(ΔSLQ)   10.6 ± 1.1 >120^(b) 0.22± 0.04 (0.95) 1.84 ± 0.15 (0.99) ^(a)Clotting times were determinedusing Pro-deficient plasma as described in the Examples inquadruplicate. ^(b)No clotting time could be detected following 120 sincubation time-period. ^(c)The rates of rPro consumption were obtainedfollowing scanning densitometry of gels studying rPro activation. Someof the gels used are shown in FIGS. 3, 4, and 5. The final rate of rProconsumption in the presence of membrane-bound fXa or prothrombinase wascalculated using the apparent first order rate constant, k (s⁻¹),obtained directly from the graph following plotting of the data asdescribed in the Examples section. ^(d)The numbers in parenthesisrepresent the goodness of fit (R²) to the equation representing firstorder exponential decay using the software Prizm.

The results shown in Table 1 demonstrate that rPro^(Δ473-487),rPro^(ΔN10), rPro^(ΔC10), and rPro^(ΔS5V) are unable to induce clottingunder the conditions described. In contrast, while, Pro^(PLASMA),rPro^(WT) and rPro^(S478A), had comparable clotting times of 12.7 s,12.2 s and 11.7 s respectively, rPro^(L480A) exhibited a minimal butsignificant prolonged PT of ˜30 s (FIG. 2). Surprisingly, whilerPro^(SL→AA) and rPro^(SQ→AA) had slow but comparable PTs of ˜30 s, thetriple mutant rPro^(SLQ→AAA) was severely ineffective in fibrin clotformation (PT ˜116 s) while rPro^(ΔSLQ) had a PT around 140 s (notshown). In contrast, rPro^(Δ473-487), rPro^(ΔN10) rPro, rPro^(ΔC10), andrPro^(ΔS5V) are unable to induce clotting under the conditionsdescribed. These functional data demonstrate that either rPro^(SLQ→AAA)cannot get activated to rIIa in a timely fashion, or that rIIa^(SLQ→AAA)formed is catalytically impaired because of the mutation, or both. Sinceprevious data has shown that the Ser⁴⁷⁸→Ala transition in IIa is of noconsequence for its chromogenic and proteolytic activity, overall theseresults demonstrate for the first time that both Leu⁴⁸⁰ and Gln⁴⁸¹ havea profound effect on IIa generation and/or IIa activity during fibrinclot formation or both.

The Activation of rPro Molecules.

To ascertain the effect of region 473-487 of prothrombin on its abilityto be activated by membrane bound fXa alone, in the absence of fVa, weassessed the pattern of activation by gel electrophoresis over a 2 htime-period (FIG. 3). Panel 3A, shows a control experiment anddemonstrates that Pro^(PLASMA) activation by membrane-bound fXa proceedsfollowing initial cleavage at Arg²⁷¹, through the intermediateprethrombin-2 with very slow gradual appearance of the B chain of IIabecause of inefficient rate of cleavage at Arg³²⁰. Astonishingly, withthe removal of amino acids 473-487 from prothrombin (FIG. 3B), there isacceleration of rPro^(Δ473-487) consumption through initial cleavage atArg²⁷¹ that is evident by the prompt appearance of prethrombin-2.Additional examinations of the intensity of the B-chain of thrombinreveal a substantially delayed cleavage at Arg³²⁰ of the recombinantdeletion mutant prothrombin molecule, compared with rPro^(WT) resultingin insignificant IIa generation. Scanning densitometry of the gels shownin FIGS. 3A and 3B showed that the rate of rPro^(Δ473-487) consumptionby membrane-bound fXa is approximately 4-fold increase compared to therate of cleavage of rPro^(PLASMA) under similar experimental conditions(Table 1). These data reveal that amino acid sequence 473-487 ofprothrombin provides a potential obstruction for efficient initialcleavage of prothrombin at Arg²⁷¹ by membrane-bound fXa alone in theabsence of fVa.

To improve our understanding of the fundamental role of amino acidregion 473-487 for Pro activation by prothrombinase, we studied thepattern of Pro activation by fully assembled prothrombinase by gelelectrophoresis over a 2 h time-period. A control experiment (FIG. 3C)demonstrates that, under the conditions used, Pro^(PLASMA) proceeds,following initial cleavage at Arg³²⁰, through the enzymatically activeintermediate meizothrombin, as confirmed by the appearance of fragment1⋅2-A. Rapid cleavage of this fragment at Arg²⁷¹ leads to the formationof IIa. In contrast, activation of rPro^(Δ473-487) under similarexperimental conditions is significantly delayed through the samepathway as verified by the late appearance of the B chain of IIa (FIG.3D). Scanning densitometry of the gels shown in FIGS. 3C and 3D showedthat rPro^(Δ473-487) is consumed with a rate that is ˜27-fold slowercompared to the rate of rPro^(PLASMA) consumption under the experimentalconditions used (Table 1). These data suggest that under conditions ofsaturating amounts of fVa with respect to fXa, amino acid sequence473-487 of Pro plays a preeminent role because it is required for fastand efficient initial cleavage of Pro at Arg³²⁰ by prothrombinase.

To further investigate the effect of the deletions and point mutationson rPro cleavage and activation by membrane-bound fXa alone, we studiedrPro activation by gel electrophoresis of all mutants detailed inFIG. 1. Panel 4A shows a control experiment and demonstrates thatrPro^(WT) activation by membrane-bound fXa proceeds typically followinginitial cleavage at Arg²⁷¹, as its plasma counterpart through theintermediate prethrombin-2 with very slow and minimal appearance of theB chain of IIa because of unproductive rate of cleavage at Arg³²⁰. Withthe removal of amino acids 478-482 from rPro^(ΔS5V) (FIG. 4B), there isacceleration of rPro^(Δ478-482) consumption by fXa alone through initialcleavage at Arg²⁷¹ that is evident by the rapid appearance ofprethrombin-2. The fact that no trace of B-chain of IIa is apparentunder the conditions employed, suggests a substantially deferred rate ofcleavage at Arg³²⁰ of the deletion mutant compared with cleavage ofrPro^(WT) resulting in insignificant amounts of IIa generation. Scanningdensitometry of similar gels shown in FIGS. 4A and 4B showed that therate of consumption of all rPro molecules by membrane-bound fXa alone isapproximately 2.3-8-fold increased compared to the rate of cleavage ofrPro^(WT) (FIG. 4C, Table 1). However, while with rPro^(S478),rPro^(L480A), rPro^(SL→AA), and rPro^(SQ→AA) minimal amounts of theB-chain of IIa are formed (not shown), when studying rPro^(ΔN10),rPro^(ΔC10), rPro^(ΔS5V), and rPro^(SLQ→AAA) activation, there isaccumulation of prothrombin-2 with no significant amounts of B chain ofIIa generated (FIG. 4B). These data confirm our findings withrPro^(Δ473-487) (FIG. 3) and reveal that the dipeptide Leu⁴⁸⁰-Gln⁴⁸¹within the fifteen amino acid stretch 473-487 of Pro appear to beresponsible for the similar effects observed with rPro^(ΔN10),rPro^(ΔC10), rPro^(ΔS5V), rPro^(SLQ→AAA) and rPro^(ΔSLQ) when studyingrPro molecules activation by membrane-bound fXa alone in the absence offVa (FIG. 4D and Table 1).

To improve our fundamental understanding of the essential role of aminoacids Leu⁴⁸⁰ and Gln⁴⁸¹ for Pro activation, we studied the pattern ofall rPro molecules activation shown in FIG. 1 by fully assembledprothrombinase (i.e. in the presence of an excess of fVa) by gelelectrophoresis over a 2 h time-period (FIG. 5). A control experiment(FIG. 5, panel A) demonstrates that under the conditions used rPro^(WT)proceeds as its plasma counterpart following initial cleavage at Arg³²⁰,through the enzymatically active intermediate meizothrombin, asconfirmed by the appearance of fragment 12-A. Rapid cleavage of thisfragment at Arg²⁷¹ leads to the formation of rIIa. Similar results werefound when using rPro^(S478A) (FIG. 5B) demonstrating that theSer⁴⁷⁸→Ala transition alone is of no consequence for timely Proactivation by prothrombinase. In contrast, activation of rPro^(ΔS5V) andrPro^(SLQ→AAA) under similar experimental conditions was significantlydelayed through the same pathway as verified by the lingering offragment 1⋅2-A at the late time points and the late appearance of the Bchain of rIIa (FIG. 5, panels C and D). Similar results were obtainedwith rPro^(ΔSLQ) (Table 1). A systematic analysis of the activation ofall rPro mutant molecules by prothrombinase using similar experimentalprocedures, followed by scanning densitometry of the gels andcalculation of the rate of rPro consumption, revealed the existence oftwo groups: a group of molecules represented by Pro^(PLASMA), rPro^(WT)and rPro^(S478A) (also containing rPro^(L480A), rPro^(SL→AA), andrPro^(SQ→AA)) that are efficiently activated by prothrombinase, and asecond group of proteins represented by rPro^(ΔS5V) and rPro^(SLQ→AAA)(including rPro^(ΔN10), rPro^(ΔC10), and rPro^(ΔSLQ)) that are activatedby fully assembled prothrombinase with a rate that is 13-17-fold slowerthan for the first group (FIG. 6A, inset and Table 1). The data suggestthat under conditions of saturating amounts of fVa with respect to fXa,the dipeptide Leu⁴⁸⁰-Gln⁴⁸¹ of prothrombin play a leading role duringPro activation because they are required for fast and efficient initialcleavage at Arg³²⁰ by prothrombinase (FIG. 6B, the deficient step isrepresented by the red arrow). It is quite remarkable that similarexperiments analyzing the activation of rPro^(ΔSLQ) by fully assembledprothrombinase performed with different reagents (fXa and PCPS vesicles)and several months apart, produced almost identical rates of activationas the rate obtained for the activation of rPro^(ΔS5V) (Table 1),attesting of the validity of our results and of the crucial role ofamino acids Leu⁴⁸⁰-Gln⁴⁸¹ for efficient rPro activation byprothrombinase.

Kinetic Analyses of rPro Molecules Activation.

In order to understand the effect of the Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹→Asubstitutions on the activity of prothrombinase in activating the rPromolecules, we first examined the rates of rIIa formation from all rPromolecules under similar experiment conditions. Historically, this methodwas designed to identify any deficiency in fVa or fXa as part ofprothrombinase in cleaving and activating Pro and is measured indirectlyby using IIa generation as a reporting probe with a chromogenicsubstrate. The comprehensive kinetic data for several mutants are shownin FIG. 7 with the kinetic constants derived directly from the fitteddata reported in Table 2.

Kinetic constants of plasma Pro and various rPro mutant moleculesactivation by prothrombinase are shown in Table 2.

TABLE 2 k_(cat)/K_(m) Prothrombin K_(m) ^(b) k_(cat) ^(b,d) (M⁻¹ · s⁻¹)· Decrease^(f) species (μM) (min⁻¹) R²/points/titrations^(e) 10⁸ (−fold)Pro^(PLASMA) 0.13 ± 0.02 1992 ± 68 0.93/30/3 2.5 — rPro^(WT)  0.11 ±0.015 1976 ± 57 0.97/20/2 2.9 — ^(a)rPro^(WT) 0.11 ± 0.02 2054 ± 840.97/10/1 3.1 — rPro^(Δ473-487) np^(c) — — — — rPro^(ΔN10) np — — — —rPro^(ΔC10) np — — — — rPro^(ΔS5V) np — — — — rPro^(S478A)  0.10 ± 0.0131764 ± 45 0.97/20/2 2.9 1.0 rPro^(L480A)  0.10 ± 0.015 1630 ± 490.96/20/2 2.7 1.1 rPro^(SL→AA) 0.12 ± 0.02 1734 ± 65 0.92/30/3 2.4 1.2rPro^(SQ→AA)  0.10 ± 0.015 1727 ± 52 0.94/30/3 2.9 1.0 rPro^(SLQ→AAA)2.3 ± 0.5  730 ± 101 0.96/36/4 0.053 55 ^(a)rPro^(ΔSLQ) 3.2 ± 1.5  128 ±34 0.96/9/1 0.0067 463 ^(a)Experiments with these two preparations ofrecombinant molecules were performed in parallel with same reagents (fXaand PCPS vesicles). The results shown are representative of fourseparate titrations with three different preparations of rPro^(ΔSLQ)compared to either rPro^(WT) or Pro^(PLASMA). ^(b)The K_(m) and k_(cat)of prothrombinase assembled with saturating concentrations ofrecombinant fVa molecules were determined as described in the Examplessection according to the Michaelis-Menten equation using the softwarePrizm from several different preparations of rPro molecules(representative experiments are shown in FIG. 7). Kinetic constants werederived directly from the fitted data. ^(c) no plot; data could not beplotted to the Michaelis-Menten equation using the software Prizm. inthe assay studying prothrombin activation using a chromogenic substrate.^(d)k_(cat) = V_(max)/[enzyme]; the enzyme concentrations ofprothrombinase (fXa-fVa complex on the membrane surface in the presenceof Ca²⁺) under the conditions employed herein was 10 pM. ^(e)R² is thegoodness of fit of the data points to the Michaelis-Menten equationusing the software Prizm. Points and titrations studied represent 10measurements/graph for all experiments (up to 4 μM plasma-derived Pro orrPro molecules) except experiments with rPro^(SLQ→AAA) (9measurements/graph, up to 2 μM prothrombin) and with rPro^(ΔSLQ) (9measurements/graph, up to 4 μM prothrombin). ^(f)The −fold decrease isthe ratio of the second order rate constant (k_(cat)/K_(m)) ofprothrombinase catalyzing rPro^(WT) activation compared to the secondorder rate constant of prothrombinase catalyzing activation of all otherrPro molecules.

The combined findings demonstrate that, while the single and doublealanine substitutions Pro mutants are activated by prothrombinasesimilarly providing comparable kinetic constants as the wild type orplasma Pro molecules surprisingly, kinetic analyses of prothrombinaseactivation of rPro^(SLQ→AAA) demonstrate a modest 2.7-fold decrease inthe k_(cat) with a concomitant and very significant 21-increase in theK_(m) of the reaction. Similar experiments studying rPro^(ΔSLQ)activation by fully assembled prothrombinase revealed a 29-fold increasein K_(m) with a concomitant 16-fold decrease in the k_(cat) of thereaction. A direct comparison between the data obtained withrPro^(SLQ→AAA) with the data obtained with rPro^(ΔSLQ) strongly suggestan important contribution of the backbone structure of the peptide bondbetween these three amino acids to efficient rPro activation byprothrombinase.

To quantify the interaction between the two sets of double mutations(Ser⁴⁷⁸/Leu⁴⁸⁰→A and Ser⁴⁷⁸/Gln⁴⁸¹→A) and to confirm their apparentsynergistic detrimental effect on prothrombinase function for activationof rPro^(SLQ→AAA), we have further calculated the difference in freeenergy of the transition state analog (ΔΔG_(int)) for the triple mutantas previously and punctiliously described by our laboratory. The largepositive value of ΔΔG_(int) (+2.4 kcal/mol) for the combination of themutations at Leu⁴⁸⁰ and Gln⁴⁸¹ together with the sizeable 55-folddecrease in the second order rate constant of prothrombinase forrPro^(SLQ→AAA) activation, signify that there is a deficiency inrecognition between prothrombinase and rPro^(SLQ→AAA). These findingssolidify our previous conclusion that these substitutions aredetrimental to the activation of rPro bearing the triple amino acidsubstitution by fully assembled prothrombinase. However, it is importantto note that it is also possible that rIIa^(SLQ→AAA) may also bedeficient in its own catalytic activity as observed with rIIa^(ΔSLQ),and the effect seen with rPro^(SLQ→AAA) activation may be likewise dueto the deficiency of rIIa in cleaving the chromogenic substrate. Thus,while we cannot yet assign the poor performance of prothrombinase incleaving rPro^(SLQ→AAA) solely to a deficiency in recognizing themutated substrate, and since the Ser⁴⁷⁸→Ala transition is of noconsequence for either rPro^(S478A) activation or rIIa^(S478A) activity,the overall data presented thus far suggest that amino acid sequenceLeu⁴⁸⁰-Gln⁴⁸¹ may have a dual effect in providing a prothrombinaserecognition site as well as for an exosite for the resulting enzymerequired for proper substrate tethering and cleavage. However, it isalso possible that these two amino acids are allosterically involved inboth prothrombinase interacting with Pro as well as the expression ofthe enzymatic activity of IIa.

Analyses of the Activity of rIIa Molecules.

While previous investigations have identified the specific amino acidresidues from Pro/IIa participating in either prothrombinase recognitionor IIa activity towards its physiological substrates respectively, fewstudies have shown that identical residues are involved in both Prorecognition by prothrombinase and IIa activity. In order to understandthe effect of the deletions/mutations on IIa activity, we furtherassessed the esterase and biological activity of all rIIa moleculesgenerated herein towards the chromogenic substrate S-2238 and thrombin'snatural substrates, fV, fVIII, and PC.

To understand the effect of the mutations on the esterase activity ofIIa, we determined the kinetic constants for the hydrolysis of S-2238 bythe rIIa molecules under steady state conditions. The data shown inTable 3 reveal that: 1) rIIa^(WT) produced under the conditionsdescribed by our laboratory has similar activity as previously foundwith other recombinant rIIa^(WT) preparations, and 2) rIIa^(S478A) hassimilar catalytic efficiency (k_(cat)/K_(m)) as rIIa^(WT) as previouslydemonstrated. In addition, we also found that while rIIa^(SLQ→AAA) wasdevoid of activity towards S-2238, rIIa^(SQ→AA) has similar esteraseactivity as rIIa^(WT), whereas rIIa^(L480A) and rIIa^(SL→AA) are themost deficient in S-2238 hydrolysis among the single and double alaninemutants when compared to rIIa^(WT) or to rIIa^(S478A) (Table 3). Thecombined data clearly demonstrates that amino acid Leu⁴⁸¹ plays animportant role during the expression of IIa chromogenic activity andthat the integrity of amino acid sequence Glu⁴⁸⁰-Leu⁴⁸¹ is required foroptimum expression of the esterase activity rIIa.

TABLE 3 k_(cat)/K_(m) α-Thrombin K_(m) ^(a) k_(cat) ^(c) (M⁻¹ · s⁻¹) ·species (μM) (s⁻¹) R^(2d) 10⁶ rIIa^(WT)  47 ± 1.8 22.7 ± 2.7 0.93 4.8rIIa^(S478A) 8.5 ± 1.5 36.5 ± 2.4 0.98 4.3 rIIa^(L480A) 8.9 ± 1.7 15.5 ±1.5 0.98 1.7 rIIa^(SL→AA) 7.7 ± 3.0 14.7 ± 2.0 0.92 1.9 rIIa^(SQ→AA)10.8 ± 4.2  33.1 ± 4.9 0.93 3.0 rIIa^(SLQ→AAA) np^(b) np np — ^(a)TheK_(m) of rIIa molecules for S-2238 was determined as described in theExamples section according to the Michaelis-Menten equation using thesoftware Prizm. Kinetic constants shown were derived directly from thefitted data. ^(b)no data could be plotted. ^(c)k_(cat) =V_(max)/[enzyme]; the Vmax was calculated as described in the Examplessection and the enzyme concentrations of rIIa was 4 nM for allexperiments shown. ^(d)R² is the goodness of fit of the data points tothe Michaelis-Menten equation using the software Prizm.

The data shown in FIGS. 8 and 9 demonstrate that while rIIa^(WT) andrIIa^(S478A) cleave and activate both cofactors with similar rates(FIGS. 8A, 8C, 9A, and 9C), rIIa^(ΔC10) and rIIa^(ΔS5V) are totallydeficient in cleaving both cofactor molecules over a three-hourincubation period (FIGS. 8B, 8H, 9B, and 9H). These data are in completeagreement with our findings shown in Table 1, explain the fact thatrPro^(ΔC10) and rPro^(ΔS5V) are devoid of clotting activity, and furtherattest of the dual role of the dipeptide Leu⁴⁸⁰-Gln⁴⁸¹ duringcoagulation. Further analyses of the single or double mutants reveal aslight differentiation in cleavage and activation of the two cofactorsby the various rIIa molecules. While rIIa^(L480A) and rIIa^(SL→AA)appear devoid of activity towards fV (FIGS. 8D and 8E), both moleculesslowly cleave fVIII at the Arg³⁷² and Arg¹⁶⁸⁹ activating cleavage sites(FIGS. 9D and 9E). Similarly, while rIIa^(SLQ→AAA) has no apparentactivity towards fV (FIG. 8G) over a three hr time-period, the mutantenzyme cleaves fVIII slowly at the non-activating Arg⁷⁴⁰ cleavage site(FIG. 9G). Finally, while rIIa^(SQ→AA) cleaves fV efficiently at Arg⁷⁰⁹to produce the heavy chain of fV and a Mr 220,000 intermediate (FIG.8F), it is also efficient in cleaving fVIII at the Arg³⁷² and Arg¹⁶⁸⁹activating cleavage sites (FIG. 9F). These two cofactors have strategicfunctions within the amplified coagulation response to vascular damageand must be activated to perform accordingly within their respectiveenzymatic complexes. The combined data explain the impaired procoagulantactivity of rPro^(SLQ→AAA) (FIG. 2) which is deficient in producinglarge amounts of rIIa in a timely fashion (FIG. 5D). However, even whenrIIa^(SLQ→AAA) is generated, the recombinant enzyme is deficient inactivating the procofactors.

We next assessed the capability of the rIIa in the presence ofthrombomodulin to activate PC and produce APC. FIG. 10 shows the resultsof such of an experiment and demonstrates that while rIIa^(ΔS5V) cannotcleave and activate PC, rIIa^(SLQ→AAA) has small but significantactivity generating minute amounts of APC (FIG. 10, lanes 8 and 9) whichin turn can cleave fV at Arg⁵⁰⁶/Arg³⁰⁶ and produce the characteristic Mr30,000 fragment (data not shown). All other rIIa mutant moleculestested, for APC generation, have similar activities as rIIa^(WT) orplasma-derived IIa under the condition described (FIG. 10).

These data demonstrate a differential requirement of IIa for cleavageand activation of both the procofactor molecules and PC and attest ofthe sensitive requirements of fV for cleavage and activation by IIa.Overall, these results provide original and novel indications that aminoacids Leu⁴⁸⁰ and Gln⁴⁸¹ within the serine protease domain of Pro, servea dual purpose and are thus required for both efficient cleavage atArg³²⁰ by prothrombinase, but also represent an obligatory exosite fortimely fV, fVIII, and PC activation. It is quite astonishing that thesetwo amino acids identified herein have such a crucial physiological dualrole during clot formation and can now be considered as the thirdexosite on Pro.

The data provided herein demonstrate that amino acid region 473-487 ofPro is required for timely activation of Pro through the meizothrombinpathway. While prior work using synthetic peptides suggested that thisregion of the cofactor may contain a fVa-dependent fXa binding site forPro, the data presented herein with recombinant Pro molecules providesfor the first time a mechanistic interpretation of these findings andidentifies the crucial amino acids from this sequence responsible forthe effect observed.

To elucidate the number and identity of the required amino acids withinamino acid sequence 473-487 of Pro, we constructed, expressed, purifiedto homogeneity, and studied several rPro molecules with deletions andpoint mutations within this important regulatory region. We firstinvestigated the effects of the fifteen amino acid deletion withrPro^(Δ473-487), followed by experiments with rPro molecules containingoverlapping deletions within this segment (rPro^(ΔN10) and rPro^(ΔC10),rPro^(ΔS5V) and rPro^(ΔSLQ)). Several rPro molecules bearing singlemutations (rPro^(S478A), and rPro^(L480A)), double mutations(rPro^(SL→AA), rPro^(SQ→AA)), and a triple mutation (rPro^(SLQ→AAA))were subsequently made. Membrane-bound fXa cleaves Pro sequentially atArg²⁷¹ followed by Arg³²⁰, forming small amounts of IIa. Under theseconditions the activation of the deletion mutants rPro^(Δ473-487),rPro^(ΔN10) rPro, rPro, rPro^(ΔS5V) and the triple and the deletionmutants (rPro^(SLQ→AAA) and rPro^(ΔSLQ)) resulted in a modest increaseof the rate of activation. In addition, activation of these five rPromolecules by fXa alone resulted in accumulation of prethrombin-2, withno apparent IIa formed. On the other hand activation of all these rPromutants by fully assembled prothrombinase is significantly delayed. Thecombined data suggest that amino acids Leu⁴⁸⁰ and Gln⁴⁸¹ within region473-487 of Pro either represent or are responsible for the presentationof a fVa-dependent site for fXa on Pro which is essential for optimalrate of cleavage at Arg³²⁰ which in turn is required for timely IIaformation at the place of vascular injury.

The autolysis loop of APC bears strong homology with the Pro sequence473-487 (chymotrypsin numbering 149D-163). Replacement of several basicamino acids from this homologous region in APC by site directedmutagenesis to alanine, demonstrated the ability of this exosite tointeract with its substrate fVa, and differentiate between the Arg⁵⁰⁶and Arg³⁰⁶ cleavage sites. Yegneswaran et al. using synthetic peptidesprovided initial evidence that sequence 473-487 of Pro is able todisrupt prothrombinase assembly only in a fVa-dependent manner. Directbinding studies with fluorescence labeled fVa demonstrated a directinteraction of the cofactor with the peptide. However, human Pro had agreater affinity for the fluorescently labeled fVa than for the peptide473-487, potentially due to the existence of other binding exosites onPro that interact with fVa. Along these lines of evidence, Chen et al.identified one of these sequences within proexosite I of prothrombincontaining basic residues Arg³⁵, Lys³⁶, Arg⁶⁷, Lys⁷⁰, Arg⁷³, Arg⁷⁵ andArg⁷⁷ (chymotrypsin numbering), which is in close spatial proximity toregion 473-487 of Pro. These investigations revealed that followingreplacement of all basic residues from proexosite I with Glu there was asignificant effect on fXa within prothrombinase when compared to fXaalone in cleaving and activating Pro, suggesting that these specificamino acids are specific fVa-dependent recognition sites for fXa on Pro.Further kinetic studies by Chen et al. using hirudin showed that thepeptide inhibited wild-type prethrombin-1 activation by prothrombinasewhereas the hirudin peptide had no inhibitory effect on the activationof the mutated zymogen lacking the basic residues in proexosite I by fXaalone. The combined studies of Yegneswaran et al. and Chen et al.suggest the requirement of both sites for optimum productive interactionof prothrombinase with Pro and timely IIa formation.

The accelerating cofactor effect that fVa has on the prothrombinasemediated activation of Pro compared to Pro activation by fXa alone hasbeen well-studied over the past 50 years but it is still not properlyunderstood and no specific molecular role has yet been assigned to thecofactor. Research with discontinuous assays using a chromogenicsubstrate for IIa revealed that when fVa is incorporated into theprothrombinase complex the resulting K_(m) of the reaction was decreasedby 100-fold (corresponding to 100-fold increase in affinity ofprothrombinase for Pro as compared to the affinity fXa alone for thesubstrate) while the catalytic efficiency (k_(cat)) of fXa was increasedby 3,000-fold resulting in a 300,000-fold overall increase in theactivity of prothrombinase (second order rate constant) for Pro comparedto the activity of fXa alone. These data demonstrated that thetwo-subunit enzyme is one of the most proficient catalysts known in thehuman body similar to several other enzymes required for survival suchas superoxide dismutase, catalase, and carbonic anhydrase. Thissignificant increase in affinity of prothrombinase for its substrate isattributed to tighter binding of the enzymatic complex to Pro becauseits localization on the membrane surface by fVa. Thus, not only does fVaenhance the reaction of Pro activation during initiation of clotting,but at the late stages of coagulation, during the propagation phase,once all 20 nM of fV physiologically available is activated, it alsopromotes the meizothrombin pathway of IIa generation (initial cleavageto Arg³²⁰ on Pro) resulting in the formation of the major enzymaticintermediate meizothrombin with demonstrated anti-coagulant activity.The longstanding hypothesis that fVa “localizes and positions” Pro in anoptimum position for efficient catalysis by fXa consistent with theclassical role of a cofactor for catalysis, was recently confirmed bycomputational studies with prothrombinase by Shim et al. These studiesdemonstrated that the acidic COOH-terminal portion of the heavy chain offVa that is contiguous to the A2 domain of fVa, is essential in itsability to interact and snare the serine protease domain of Pro thusrepositioning the Arg³²⁰ cleavage site at an optimum position for timelycleavage by fXa and Pro activation at the site of vascular injury asearlier suggested and more recently experimentally demonstrated by ourlaboratory with synthetic peptides and recombinant fVa molecules mutatedat these specific sites.

We show that following removal of the amino acid sequence 473-487 fromPro, prothrombinase loses the ability to efficiently form IIa because ofimpaired fVa-dependent cleavage of Pro by fXa at Arg³²⁰. One easyexplanation of these results was that elimination of such a huge portionof the molecule results in significant structural changes of themolecule that in turn have deleterious effects on Pro molecularconformation resulting in deficient prothrombinase activity. In spite ofthe fact that rPro^(Δ473-487) was activated following the same pathwaysas rPro^(WT) in the presence or absence of fVa albeit with differentrates, and in the absence of a crystal structure of rPro^(Δ473-487),there was still doubt about the structural integrity and function of amolecule bearing such a large deletion. Experiments using more modestoverlapping deletions (with rPro^(ΔN10), rPro^(ΔC10), and rPro^(ΔS5V))as well as with a triple alanine mutant (rPro^(SLQ→AAA)) and a tripledeletion mutant (rPro^(ΔSLQ)), demonstrated that these molecules arealso hindered in their fVa-dependent cleavage at Arg³²⁰ to a similarlevel as rPro^(Δ473-487) (Table 1). These data provide original andunexpected evidence demonstrating that the minimal sequence required forthe 3,000-fold increase in the catalytic efficiency of prothrombinase iscarried at least partially by amino acid sequence Leu⁴⁸⁰-Gln⁴⁸¹ of Pro.The findings presented herein silence the notion that the effect seenwith rPro^(Δ473-487) may be due to a structural change of the mutantmolecule rather than to specific amino acid(s) missing from therPro^(Δ473-487), and assign the remarkable delay in Pro activation totwo specific amino acids.

The kinetic findings presented herein revealed comparable K_(m) andk_(cat) constants for prothrombinase when rPro molecules bearing thesingle and double alanine mutations were used as substrate. However,when rPro^(SLQ→AAA) was the substrate for prothrombinase in the samediscontinuous assay, there was an astonishing 21-fold increase in theK_(m) and a modest 2.7-fold decrease in the k_(cat) of the enzyme.Similar results were obtained with rPro^(ΔSLQ). Furthermore,rPro^(SLQ→AAA) and rPro^(ΔSLQ) were also found to be substantiallydeficient in clot formation in an assay using Pro-deficient plasma,while rIIa^(SLQ→AAA) was also deficient in S-2238 hydrolysis.rPro^(SLQ→AAA) was also impaired in cleaving fV, fVIII and PC. Thesedata dovetail nicely with results obtained with rIIa^(ΔS5V) andrIIa^(Δ473-487). We can thus hypothesize that the substantial increasein the K_(m) of prothrombinase towards rPro^(SLQ→AAA) is due to adeficiency in prothrombinase in recognizing the mutant molecule becauseof the lack of Leu⁴⁸⁰-Gln⁴⁸¹, while the decrease in enzymatic activityof the resulting rIIa^(SLQ→AAA) molecule is also the result of theabsence of these two important amino acids' side chain. Additional datawith rPro^(ΔSLQ) provides further evidence of the crucial role of aminoacids Leu⁴⁸⁰-Gln⁴⁸¹, and the peptide bond between these two amino acidssince, when these residues are completely eliminated, the K_(m) of thereaction increases by 32-fold while the k_(cat) decreases by a stunning16-fold (Table 2). Keeping in mind that the Ser⁴⁷⁸→Ala substitution isof no consequence on both rPro activation and rIIa function, thesesurprising and unexpected results provide strong evidence in favor ofthe dual role of amino acids Leu⁴⁸⁰ and Gln⁴⁸¹. Namely, these aminoacids are required by prothrombinase to efficiently promote cleavage ofPro at Arg³²⁰ and are also required by IIa for optimum esterase activityas well as to proficiently cleave and activate fV, fVIII, and PC.Finally, the possibility that elimination of these two residues fromrPro results in an allosteric transition of the amino acidsaround/within the active site of rIIa, thus modifying the criticaldistances between the specific residues of the catalytic triad resultingin impaired catalysis, cannot be eliminated.

A comparison of crystal structures of Pro, meizothrombin, IIa,prethrombin-1, and prethrombin-2 was carried out to identify structuraldifferences in/near the Gly⁴⁷³-Ile⁴⁸⁷ segment comprising thefVa-dependent fXa binding site. These residues adopt similarconformations in all of the crystal structures, with the NH₂-terminalresidues Gly⁴⁷³-Gln⁴⁷⁶ being quite solvent-accessible or flexible, andresidues Pro⁴⁷⁷-Ile⁴⁸⁷ being variable in their degree of solventexposure. Residue Ile⁴⁸⁷ is significantly more exposed in prothrombin(accessible surface area of >30 Å² compared to <10 Å² in meizothrombinand thrombin), as well as the adjacent Pro⁴⁸⁶ (accessible surface areaof ˜15-30 Å² reducing to <10 Å² in meizothrombin and thrombin). Theamount of solvent exposure of Ile⁴⁸⁷ and Pro⁴⁸⁶ appears to be heavilyinfluenced by the flanking loops encompassing residues Ala⁴⁴⁶-Tyr⁴⁵⁴ andLys⁵¹¹-Ser⁵²⁵ which adopt different conformations upon Pro activation(FIG. 11). For example, residues Leu⁴⁵-Gly⁴⁵³ shift by as much as 10 Åcloser to Pro⁴⁸⁶ in meizothrombin and IIa compared to Pro, partiallyshielding this residue from solvent in the meizothrombin and IIastructures. Recently, Pozzi et al. used the crystal structure ofGla-domainless Pro with active site Ser⁵²⁵→Ala to demonstrate that fVahas recognition sites in close proximity to Arg³²⁰ (Arg¹⁵ chymotrypsinnumbering). These sites create a strong electrostatic potential due to anumber of basic residues described by Chen et al. Through analysis ofthis published crystal structure, we have located this basic region tobe in the vicinity of the Leu⁴⁸⁰-Gln⁴⁸¹ amino acid sequence of Pro thatwe found to be required for efficient initial cleavage at Arg³²⁰ byprothrombinase. It is noteworthy that a very recent study by Pozzi etal. demonstrated a crucial role for linker 2 for the rate of activationof Pro by prothrombinase and suggested that this region maybe involvedin the interaction of Pro with the cofactor. These data are in completeaccord with data showing that fragment 1 and more precisely the kringle2 region is involved in the interaction of fVa as part of prothrombinasewith Pro. Finally, a close comparison of crystal structures of Pro andIIa revealed that residues Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹ adopt similarconformations in both structures. The Ser⁴⁷⁸ side chain is exposed onthe surface of both molecules, while the Leu⁴⁸⁰ side chain is surroundedby other residues and not accessible to solvent. Gln⁴⁸¹ is partiallysolvent-exposed in both Pro and IIa. The Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹ residuesare near ABE-I (FIG. 11) but >15 Å from the catalytic Ser⁵²⁵ residue,and even more distant from ABE-II.

In conclusion, in this study we provide original and unequivocalevidence for the dual effect of amino acids Leu⁴⁸⁰ and Gln⁴⁸¹ of Pro.This study also provides unprecedented information on prothrombinase andsheds light into the understanding of the “cofactor effect” of fVa andthe precise molecular interactions that fVa has with a minimal regionlocated in the serine protease domain of Pro. In addition, through theuse of alanine substitution in exposed amino acids (Ser⁴⁷⁸ and Gln⁴⁸¹)or homologous to other serine proteases (Leu⁴⁸⁰) (FIG. 11), we canconclude that the two valines (Val⁴⁷⁹ and Val⁴⁸²) in this region, whichare held constant in all of our mutant molecules bearing alaninesubstitutions, or in the rPro^(ΔSLQ) molecules, do not appear tocontribute significantly to the overall molecular interactions betweenthe fVa-dependent interaction of fXa and rPro or in the overallcatalytic and esterase activity of rIIa. Future mutagenesis studieswithin the amino acids uncovered herein, paired with judiciouslyselected mutations within proexosite-I and/or proexosite-II of Pro,should be able to elucidate the intermolecular communications withinPro, required for both optimal fVa-dependent activation of Pro andsubsequent IIa catalytic activity towards its numerous physiologicalsubstrates. Finally, our results provide evidence for the production oflarge quantities of rPro^(ΔS5V), rPro^(SLQ→AAA), or rPro^(ΔSLQ) thatcould be used as therapeutic agents since they would compete with thenatural substrate in vivo, when infused in individuals withprothrombotic tendencies.

All references to singular characteristics or limitations of the presentdisclosure shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

While embodiments of the invention have been illustrated and described,it is not intended that these embodiments illustrate and describe allpossible forms of the invention. Those familiar with the art to whichthe invention relates will appreciate other ways of carrying out theinvention defined by the following claims. Rather, the words used hereinare words of description and not limitation, and it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theinvention as set forth herein.

ABBREVIATIONS USED ARE

-   PS, L-α-phosphatidylserine; PC, L-α-phosphatidylcholine;-   PCPS, small unilamellar phospholipids vesicles composed of 75% PC    and 25% PS (w/w);-   SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis;-   rPro^(WT), recombinant wild type human prothrombin;-   rIIa^(WT) recombinant wild type human α-thrombin;-   rPro^(Δ473487), recombinant human prothrombin with region 473-487    deleted;-   rIIa^(Δ473-487), recombinant human α-thrombin with region 473-487    deleted;-   rPro^(ΔN10) recombinant human prothrombin missing amino acids    GKGQPSVLQV;-   rIIa^(ΔN10) recombinant human α-thrombin missing amino acids    GKGQPSVLQV;-   rPro^(ΔC10) recombinant human prothrombin missing amino acids    SVLQVVNLPI;-   rIIa^(ΔC10) recombinant human prothrombin missing amino acids    SVLQVVNLPI;-   rPro^(ΔS5V), recombinant human prothrombin with region SVLQV    deleted;-   rIIa^(ΔS5V), recombinant human α-thrombin with region SVLQV deleted;-   rPro^(S478A), recombinant human prothrombin with the mutation    S⁴⁷⁸→Ala;-   rIIa^(S478A), recombinant human α-thrombin with the mutation    Ser⁴⁷⁸→Ala;-   rPro^(L480A), recombinant human prothrombin with the mutation    Leu⁴⁸⁰→Ala;-   rIIa^(L480A), recombinant human α-thrombin with the mutation    Leu⁴⁸⁰→Ala;-   rPro^(SL→AA), recombinant human prothrombin with the mutation    Ser⁴⁷⁸/Leu⁴⁸⁰→Ala;-   rIIa^(SL→AA), recombinant human α-thrombin with the mutation    Ser⁴⁷⁸/Leu⁴⁸⁰→Ala;-   rPro^(SQ→AA), recombinant human prothrombin with the mutation    Ser⁴⁷⁸/Gln⁴⁸¹→Ala;-   rIIa^(SQ→AA), recombinant human α-thrombin with the mutation    Ser⁴⁷⁸/Gln⁴⁸¹→Ala;-   rPro^(SLQ→AA), recombinant human prothrombin with the mutation    Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹→A;-   rIIa^(SLQ→AA), recombinant human α-thrombin with the mutation    Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹→A;-   rPro^(ΔSLQ), recombinant human prothrombin with amino acids    Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸ deleted;-   rIIa^(ΔSLQ), recombinant human α-thrombin with amino acids    Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹ deleted.

What is claimed is:
 1. A recombinant prothrombin polypeptide, whereinthe polypeptide comprises a deletion of amino acid residues Ser⁴⁷⁸,Leu⁴⁸⁰, and Gln⁴⁸¹ or a substitution of Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹ withalanine.
 2. The recombinant prothrombin of polypeptide claim 1, whereinthe polypeptide comprises a substitution of amino acid residues Ser⁴⁷⁸,Leu⁴⁸⁰, and Gln⁴⁸¹ with alanine.
 3. The recombinant prothrombin ofpolypeptide claim 1, wherein the polypeptide comprises a deletion ofamino acid residues Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹.
 4. The recombinantprothrombin of polypeptide claim 1, wherein the modification is asubstitution of Ala for each of the Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹.
 5. Amethod of reducing coagulation in a subject who has or is at risk ofhaving thrombosis, the method comprising administering a polypeptideaccording to any one of claims 1 and 2-3.
 6. An isolated polynucleotidewhich encodes a polypeptide characterized by: (a) having anti-coagulantactivity; and (b) having the amino acid sequence of prothrombincomprising a deletion of amino acid residues Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹or a substitution of Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹ with alanine.
 7. Anon-human host cell which contains the polynucleotide of claim
 6. 8. Arecombinant expression vector which contains the polynucleotide of claim6.
 9. The vector of claim 8, wherein the vector is a plasmid.
 10. Thevector of claim 8, wherein the vector is a virus.
 11. A method forproducing a polypeptide having an amino acid sequence of prothrombin,wherein the polypeptide acts as a prothrombin molecule which isincapable of activation comprising: (a) introducing into a host cell anexpression vector which contains a nucleotide sequence which encodes apolypeptide having an amino acid sequence of prothrombin comprising adeletion of amino acid residues Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹ or asubstitution of Ser⁴⁷⁸, Leu⁴⁸⁰, and Gln⁴⁸¹ with alanine; (b) culturingthe host cell in an appropriate medium; and (c) isolating thepolypeptide product encoded by the expression vector.